1. Cell Cycle Regulator and Tumor Development
Lee Young Kyung, M.D.

2. Cell Cycle Mitosis RNA and protein synthesis G0 phase Reactivation
6-12 hrs
2n DNA
S phase
6-8 hrs
DNA synthesis
2n-4n DNA
G2 phase
3-4 hrs
4n DNA
1 hr M
G0 phase
Reactivation
Indefinite withdrawal
RNA and protein
synthesis
Mitosis
Cell cycle : between two mitotic divisions
Interphase : from the end of one mitosis to the start of the next
M phase : actual division, visible mitosis
G1-S-G2-M (S means synthesis, G means gap with no DNA synthesis)
G1 : RNA and protein synthesis, no DNA replication
S : DNA replication (2n-4n)
G2 : two complete diploid sets of chromosomes
Control point : G1-S, G2-M

3. cdk-cyclin complexes cdk cyclin G2/M phase G1/S phase
Regulator for G1/S phase or G2/M phase
Dimer : cdk - cyclin dependent kinase
cyclin - regulatory domain
Phosphorylation & dephosphorylation
cdk
cyclin
G2/M phase G1/S phase
P cdk2, cdk4
cyclin A, B1, B2 cyclin A, D1, D2, D3, E

4. cdk inhibitors RB RB G0/G1 transition E2F cdk 4, 6 cyclin D1,2,3 E2F
P15/p16 (ink4)
P21(cip1/WAF1)
P27(Kip1) RB
E2F
cdk 4, 6
cyclin
D1,2,3 P RB P
E2F
G0/G1 transition

5. Retinoblastoma protein (pRb)
Retinoblastoma : loss of Rb protein function
Rb gene : chromosome 13q14
Rb protein : nuclear phosphoprotein
Not phosphorylated in resting cells : binding E2F
phosphorylated during cell cycle by cdk/cyclin complexes
Overexpression of Rb : prevent cell proliferation
Related tumor suppressors : p16, p21, cyclin D

6. p16INK4a First noted by Xiong et al. in 1993
16kD, CDK inhibitor tumor suppressor
Related to CDK4, 6 & cyclin D
Choromosome 9p21 : MTS1 = CDKI = CDKN2 = INK4
(INK4a-d ; CDKN2A-D)
INK4 family members : p16INK4a, p15INK4b, p18INK4c, p19INK4d

7. Functions of p16INK4a
Binding directly to CDK4, 6 & preventing association with cyclin D
Inhibit the activity of preformed cylclin D/CDK4,6 complex
“G1 cell cycle arrest”
Low expression in primary tissues
Role in replicative senescence
Need normal Rb function
Inverse correlation with pRb level

8. p14ARF protein p16INK4A protein
CDKN2A
gene
p14ARF mRNA
p16INK4A mRNA
E1β
E1α E2 E3

9. p14ARF Alternative product of INK4a/CDKN2A gene locus :
β transcript : exon 1β spliced to exon 2
Using different promotor and alternative reading frame
G1/G2 arrest
Related to p53 – mdm2

10. p53 mdm2 Loss of functions develop tumor. :
by missense mutation of one allele
by deletion of both allele
Activate
transcription
Bind to DNA
Oligomerization
Bind to damaged DNA
mdm2

11. p53 & mdm2 Mdm2 : destabilize & inhibit p53 by binding N-terminal
P53 activates mdm2 transcription
“negative feedback”
p14ARF – p53 - mdm2
Increase p53 expression p21cip1 & mdm2 expression
protect p53 degradation by mdm2
negatively regulated by p53

12. Location of primers Sequence of primers V6 VA p16INK4A exon 1α Exon 2
p14ARF exon 1β
Exon 2
Exon 3
Sequence of primers
Primer Sequence
V6 5' GCT GCC CAA CGC ACC GAA TA 3'
V9 5' CCC TCG TGC TGA TGC TAC TGA 3'
VA 5' ACC ACC AGC GTG TCC AGG AA 3'

13. Table 1. p16INK4A and p14ARF inexpression patterns in
hematologic malignancy patients
1 AML, M1 normal normal
2 AML, M1 normal normal
3 AML, M2 normal normal
4 AML, M3 normal normal
5 AML, M3 normal normal
6 AML, M4e normal normal
7 AML, M6, relapse increased increased
1 ALL, L1 normal normal
2 ALL, L1 increased normal
3 ALL, L1 (T ) increased normal
4 ABL, persistent normal normal
1 MDS, RA increased increased
2 MDS, RAEB normal normal
3 MDS, RAEB increased normal
4 MDS, RAEB normal increased
5 MDS, RAEB in t normal normal
1 CML, chronic increased increased
2 CML, chronic increased increased
3 CML, chronic increased normal
4 CML, chronic increased normal
5 CML, chronic normal increased

14. Table 2. Overexpressions of p16INK4A and p14ARF in hematologic
malignancy
Disease Total number p16INK4A mRNA p14ARF mRNA
of cases overexpression overexpression
Normal
AML (17%) (17%)
ALL 4 2*(40%) (0%)
MDS (40%) (40%)
CML 5 4†(80%) †(60%)
*P<0.1 †P<0.05

15. P16INK4A P14ARF Fig. 3. RT-PCR result of p16INK4A and p14ARF mRNAs.2 3 HL
P16INK4A
267 bp
234 bp
213 bp 192 bp
184 bp
P14ARF M 1 2 3 HL
267 bp
234 bp
213 bp 192 bp
184 bp
Fig. 3. RT-PCR result of p16INK4A and p14ARF mRNAs.
Lane 1 : CML patient. Both mRNA expressions increased.
Lane 2 : MDS patient, only p16INK4A expressions increased.
Lane 3 : Normal bone marrow, faint or barely looking both mRNA.
M : Marker V (Boehringer Mannheim, Germany), HL : Hela cell line